Oecologia (1999) 120:123±131
Ó Springer-Verlag 1999
Alan C. Gange á Erica Bower á Valerie K. Brown
Positive effects of an arbuscular mycorrhizal fungus on aphid life history traits
Received: 1 February 1999 / Accepted: 22 March 1999
Abstract Two generations of two aphid species (Myzus ascalonicus and M. persicae) were reared on Plantago lanceolata plants, with and without root colonization by the arbuscular mycorrhizal fungus, Glomus intraradices. Life history traits of the aphids measured were nymphal development time, teneral adult weight, growth rate, total fecundity, adult longevity and duration of postreproductive life. For both aphids in both generations, mycorrhizal colonization increased aphid weight and fecundity, while other traits were unaected. The increases were consistent between generations. In a second experiment, M. persicae was reared on plants with and without the fungus, under varying N and P regimes. The results of N addition were inconclusive because there was high aphid mortality. However, under P supplementation, positive eects of the mycorrhiza on aphid growth were seen at low and medium P levels, while at high P levels these eects disappeared. The positive effects of mycorrhizal colonization reported here are contrary to the majority of previous studies with chewing insects, which have reported negative eects. A number of possible mechanisms for this apparent discrepancy are discussed. Key words Arbuscular mycorrhiza á Aphid á Insect Phosphate
Introduction Arbuscular mycorrhizal (AM) fungi associate with the majority (often over 90%) of plant species in natural A.C. Gange (&) á E. Bower School of Biological Sciences, Royal Holloway University of London, Egham Hill, Egham, Surrey, TW20 0EX, UK e-mail:
[email protected], Fax: +44-1784-470756 V.K. Brown CABI Bioscience Environment UK Centre (Ascot), Silwood Park, Buckhurst Road, Ascot, Berks., SL5 7TA, UK
ecosystems (Brundrett 1991). Plant growth has often been shown to be enhanced by this association, mainly due to an increased ability to take up nutrients, principally nitrate (e.g. Tobar et al. 1994) and phosphate (reviewed in Smith and Read 1997). However, AM fungi may also aect positively many other aspects of plant biology. For example, these fungi have been shown to enhance drought resistance (e.g. Davies et al. 1992), competitive ability (e.g. West 1997a), and resistance to fungal pathogens (West 1997b) and insect herbivores (Gange and Bower 1997). With so many positive eects on plant performance, it is often unclear as to what negative feedback system may regulate the stability of the mutualistic relation (Allen 1991). Recently, several studies have shown that AM fungi can have signi®cant interactions with insect herbivores. The ®rst example was provided by Rabin and Pacovsky (1985), who showed that growth of two generalist chewing insects (Heliothis zea Boddie and Spodoptera frugiperda J.E. Smith) was reduced on leaves from soybean plants colonized with the mycorrhizal fungus Glomus fasciculatum Thaxt. sensu Gerd., compared with uncolonized plants. More recently, Gange and West (1994) found that reducing mycorrhizal levels of ®eldgrown Plantago lanceolata L. resulted in higher levels of damage by chewing insects, while a laboratory trial demonstrated that performance of the generalist chewing insect Arctia caja L. was lower on highly mycorrhizal plants. Meanwhile, Gange and Nice (1997) demonstrated that the performance of the thistle gall ¯y, Urophora cardui (L.) Scop., was increased if natural levels of mycorrhizal colonization of its host plant were reduced by fungicide application. Root-feeding insects may also be negatively aected by AM fungi, as Gange et al. (1994) found that survival and growth of black vine weevil, Otiorhynchus sulcatus (Fab.), were halved when larvae were fed on plants of Taraxacum ocinale Weber, colonized by G. mosseae (Nicol and Gerd). Not all insect-mycorrhizal interactions may be detrimental to the insect. In the study of Pacovsky et al. (1985), reproduction of the foliar-feeding aphid Schi-
124
zaphis graminum on sorghum was unaected by mycorrhizal colonization. Positive eects of mycorrhizas on insect performance have also been recorded. In a small part of the study by Gange and West (1994), growth of a sap-feeding aphid Myzus persicae Sulzer was examined on P. lanceolata plants with natural and reduced levels of AM colonization. In this case, aphid weight and embryo content were increased when mycorrhizas were present. A further positive interaction was reported by Borowicz (1997), where AM colonization of soybean (Glycine max (L.) Merr.) roots increased the survival and mass at pupation of Mexican bean beetle (Epilachna varivestis Mulsant) larvae. However, this eect was only seen at low P levels, and when P was plentiful, no eect on the insects was observed. The study of Borowicz (1997) highlights a key issue in attempting to understand these interactions, namely that AM fungi can alter the nutrient status of a plant and thereby food quality and/or resistance to insect herbivores. As Borowicz (1997) points out, insect herbivore performance responses to plant nutrient stress are often non-linear, with maximized performance at intermediate plant stress (e.g. Mattson and Haack 1987). Therefore, negative, null or positive eects of AM fungi on insects are possible, depending on the degree of alleviation of nutrient stress by the fungus. However, the eect of AM fungi on the nutrient status of a plant is far from clear. Many studies have shown that AM colonization increases P uptake and thus shoot P levels (Bolan 1991), but few have shown N uptake, and even fewer, changes in foliar tissue N concentration (Smith and Read 1997). Indeed, in the two previous herbivore/mycorrhizal studies, Gange and West (1994) and Gange and Nice (1997) recorded an increase in foliar tissue total N when mycorrhizas were reduced by fungicide. To date, all the insect/mycorrhizal studies have measured just one or two insect performance parameters in the presence/absence of the fungi. In this paper, our aim is to report on a range of life history traits of two sap-feeding insects, in order to obtain a clear idea of which insect attributes are aected by AM colonization. We use the same insect (M. persicae) as Gange and West (1994) in order to provide comparability, and include another closely related species, M. ascalonicus Doncaster. Both species were reared on P. lanceolata, a common host for both insects in the ®eld. The aphids were reared through two complete generations to examine consistency of responses over time. Furthermore, we report a further experiment, aimed at determining whether the interactions between M. persicae and AM fungi are mediated by varying soil nutrient levels (N and P).
Materials and methods Comparison of M. ascalonicus and M. persicae An identical procedure was used for rearing the two aphid species. For each species, 24 2-week-old seedlings of P. lanceolata (germi-
nated in sterile sand) were planted into 13-cm-diameter pots, ®lled with John Innes number 2 compost (Gem Gardening). Immediately before planting, mycorrhizal inoculum was added to half of the pots by incorporating 1.5 g of inert clay granules containing hyphae and spores of Glomus intraradices (Vaminoc, MicroBio, Hemel Hempstead). This amount of inoculum had an infectivity of 302 39.4 fungal propagules g)1 (measured by the most-probable-number method). The other half of the pots received granules sterilized by gamma irradiation, as this provides the least amount of nutrient addition and controls for the eect of granule addition. Plants were grown in a constant-temperature room at 20°C and light regime of 16:8 L:D for 12 weeks. No extra nutrients were added, but plants were watered three times a week with 50 ml of distilled water. To check that the mycorrhiza had colonized the plants, 12 weeks after inoculation, a 5-mm sterile cork borer was used to remove a plug of soil containing roots from each pot. The hole was ®lled with compost and the roots washed free from each core. To check for mycorrhizal colonization they were placed on a microscope slide and examined at ´200 using a Zeiss Axiophott epi¯uorescence microscope ®tted with a UV lamp and ®lters giving a transmission of 455±490 nm blue. Under these conditions, the arbuscules ¯uoresce (Ames et al. 1982) and it has recently been shown that this is a more reliable and consistent method of mycorrhizal visualization than conventional staining methods (Gange et al., 1999). At the 12-week stage, all inoculated plants showed a degree of colonization, while no mycorrhiza was found in any plant given irradiated inoculum (hereafter referred to as `control' plants). A single apterous adult female aphid was then placed on a randomly selected mature leaf on each of the 24 plants (12 mycorrhizal and 12 non-mycorrhizal) and enclosed within a 2-cm-diameter clip cage (Noble 1958). Cages were examined daily, and when the aphid had reproduced, all aphids, except for the youngest nymph, were removed. The single nymph was left undisturbed in the cage and examined daily to check for survival and growth. No aphids died or escaped from the cages. As soon as the aphid had moulted to an adult, it was weighed on a microbalance. Thereafter, the cage was examined daily, and any ospring produced counted and removed. This procedure was continued until the adult aphid died. In this way, we were able to obtain measurements of development time to adult, adult teneral weight, adult longevity, lifetime fecundity and duration of post-reproductive life. A sample of 20 young aphids, each less than 12 h old, was weighed and the mean weight calculated. This ®gure was used as an estimate of birth weight, and to calculate the relative growth rate, following the formula given by Radford (1967). When all adult aphids of the ®rst generation had died, the experiment was repeated, on the same plants, using apterous adult females taken from an uncrowded stock culture to initiate the experiment. All the aphids reared of both species in both experiments were apterous. At the end of the second generation, all plants were harvested, the roots washed free of soil and subjected to auto¯uorescence microscopy as above. Mycorrhizal colonization was quanti®ed with the cross-hair eyepiece method of McGonigle et al. (1990). Nutrient availability and growth of M. persicae As commercial composts can vary widely in their nutrient availability, the medium used to grow plants was Seramis (Pedigree Petfoods, Melton Mowbray), which consists of inert, nutrient-free expanded clay granules. One hundred and twenty plastic pots (volume 250 ml) were ®lled with Seramis and a 0.5 g G. intraradices inoculum was placed in a layer 2 cm below the surface. This was designed to give the same amount of fungal inoculum per unit of medium as the compost in the ®rst experiment. Irradiated inoculum was added to the control plants. Macronutrient and micronutrient solutions were prepared following the mixtures given in Bower (1997), to produce three levels of N and P addition. For the nitrogen treatments, N:P:K molar ratios were 9.0:0.15:1 for the high N, 1.8:0.15:1 for the medium N and 0.36:0.15:1 for the low N. For the P treatments these were 1.8:1:1 for high P, 1.8:0.15:1 for
125 Table 1 Means, with standard errors in parentheses, of three life history traits (in days) of Myzus persicae and M. ascalonicus in two generations, when reared on mycorrhizal and non-mycorrhizal Life history parameter
Development time Longevity Post-reproductive life
Plantago lanceolata. The only signi®cant eect was found with the longevity of M. persicae in generation 1 (Mann-Whitney U = 35.5, P < 0.05)
M. ascalonicus Generation 1
M. persicae Generation 2
Generation 1
Generation 2
Nonmycorrhizal
Mycorrhizal NonMycorrhizal Nonmycorrhizal mycorrhizal
Mycorrhizal
Nonmycorrhizal
Mycorrhizal
25.0 (1.7) 31.7 (2.9) 2.4 (0.6)
22.7 (2.1) 38.7 (4.3) 2.6 (0.7)
23.4 (1.8) 28.0 (2.2) 1.3 (0.2)
25.7 (2.5) 28.2 (2.6) 1.4 (0.2)
26.5 (2.0) 30.3 (2.9) 1.8 (0.3)
23.4 (2.2) 48.7 (5.2) 2.8 (0.5)
21.5 (1.6) 56.4 (5.7) 2.3 (0.3)
medium P and 1.8:0.01:1 for the low-P solution. The P level in the N addition treatments was therefore the equivalent of that in the medium P treatment and vice versa. A single seedling (three true leaves) of P. lanceolata was planted into each pot on the day that medium and inoculum were added. Each pot was then watered with 50 ml of the appropriate nutrient solution plus 20 ml of distilled water, and the pot weight was recorded. Thereafter, plants were fed with 6 ml of the appropriate nutrient solution weekly, and distilled water was added as necessary to maintain pots at a constant weight. There were twelve treatments (three levels of two nutrients, with and without mycorrhiza) and each was replicated ten times. After 12 weeks, an adult apterous M. persicae was placed on each plant and the resulting ospring reared to adulthood as described in the ®rst experiment. In this study, aphids were weighed as soon as they were adult (teneral weight), and then dissected and the number of embryos counted as a measure of potential fecundity. The total number of embryos and those in an advanced stage of development (with pigmented eyes) were recorded. Aphid growth rates were calculated in the same manner as described above.
27.4 (2.7) 20.9 (2.7) 1.7 (0.4)
ence between generations for either species. Therefore, because of the constant temperature environment, all aphids took about the same time to mature, whether they were reared on mycorrhizal plants or not. Mycorrhizal presence caused signi®cant increases in teneral adult weight of both species in both generations (Fig. 1, Table 2). This eect appeared to be weaker in M. ascalonicus, as in generation 1, weight was increased by 20%, while in generation 2 it was increased by 28%. In M. persicae, weight was doubled by the presence of the AM fungus in generation 1, and increased by 70% in
Statistical analysis All data sets were tested for normality and homogeneity of variances. Where the assumptions of normality were met, dierences between treatments for each aphid parameter in each species were examined with the t-test. If normality was not met, the MannWhitney test was used instead. To examine any dierences between generations of aphids reared on the same plant, paired comparisons (paired t-test or Wilcoxon matched-pairs test) were employed. In experiment 2, the eect of mycorrhizal and nutrient addition and the interactions between them were analysed with two-factor analysis of variance. Separation of pairs of means was performed with linear contrasts (Sokal and Rohlf 1995). In this experiment, percentage data were subjected to angular transformation before analysis.
Results Comparison of M. ascalonicus and M. persicae No mycorrhizal colonization was detected in any of the control plants. The mean percent of root length colonized (arbuscules only) in the plants on which M. ascalonicus was reared was 13.8 2.7% (range 6.7± 20.4%) and for plants with M. persicae 14.8 4.7% (range 9.6±21.7%). There was no signi®cant eect of AM fungal presence on development time of either aphid species in each generation (Table 1). Furthermore, there was no dier-
Fig. 1 Adult teneral weight of two aphid species, reared on Plantago lanceolata, with and without colonization by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. Error bars indicate 1 SE. A Myzus ascalonicus. B Myzus persicae
126 Table 2 Results of t-test or Mann-Whitney test for the eect of mycorrhizal colonization on life history traits of M. ascalonicus and M. persicae in two generations. Mann-Whitney test results are given in italics. All n = 12 (NS not signi®cant at P = 0.05) Life history parameter
Teneral weight Growth rate Fecundity
M. ascalonicus Generation 1
M. persicae Generation 2
Generation 1
Generation 2
Statistic
P
Statistic
P
Statistic
P
Statistic
P
2.29 1.796 4.529
0.031 NS
